Infection and innate immunity in the female genital tract

Professor Martin Sheldon FRCVS

Reproductive Immunology

ROME September 2016

Farmers and veterinarians are rising to the grand challenge to feed the world

1945 2015

But there is a problem: uterine infections increasingly cause endemic disease and reduce productivity

Bacterial infection of the uterus after parturition affects all dairy cattle

40% develop clinical disease

Animals remain infertile even after successful treatment of the disease

€1.4 billion/year in EU $0.6 billion/year in USA

Bacteria cause uterine disease

Widely accepted pathogens Griffin et al 1974 Therio 1, 91 Dohmen et al 1995 Therio 43, 1379

Sheldon et al 2002 Reproduction 123, 837 Wagener et al 2015 Vet Micro 175, 286

Gram-negative Escherichia coli

Gram-positive Trueperella pyogenes

Anaerobes Prevotella spp

Fusobacterium spp

Complex uterine microbiome

Jeon et al 2015 Appl Environ Microbiol 81:6324

4

Fertility

Normal

Sub-fertile

Infertile

Sheldon et al 2009 Biol Reprod 81:1025

Post partum uterine bacterial infection and disease is common in dairy cattle

0 10 20 30 40 50 60 700

20

40

60

80

100

Infection

Perc

enta

ge o

f ani

mal

s

Days post partum

Endometritis

Normal endometrium

Metritis

Subclinical endometritis

David Noakes

The mechanisms of inflammation and defence in the bovine endometrium - a missing component?

1. What evidence is there for the role of innate immunity in the postpartum uterus?

2. What factors might scale the inflammatory response in the endometrium?

3. What impact does uterine infection have on ovarian function?

Jules Hoffmann Bruce Beutler

Nobel Prize in Physiology or Medicine 2011 awarded for discoveries concerning Toll and Toll-like receptors (TLRs) for the activation of innate immunity by pathogen-associated molecular patterns

Innate immunity depends on cellular receptors that bind pathogen-associated molecular patterns

Macrophage Neutrophil

Cytokines IL-1β IL-6 IL-10 Chemokines IL-8 CCL5 Prostaglandins PLA2 PGE2 Antimicrobial peptides LAP TAP

TLR4

LPS E. coli

Attract and activate more macrophages

and neutrophils

Pathogen clearance

Hematopoietic innate immune cells sense pathogen-associated molecular patterns

NFκB ERK p38

CD14 MD2

TLR4

8

Inflammatory gene expression

Bacterial infection of the uterus causes disease with inflammation and damage of the endometrium

Innate immunity TLR4

Prostaglandins PLA2

Cytokines IL1B IL6

Chemokines IL8 CCL5

Antimicrobial peptides

Clinical disease Histopathology Gene transcription

Inflammation - neutrophils - macrophages - damage

Evidence for innate immune responses - in vivo acute phase protein response

4 8 1 2 1 6 2 0 2 4 2 8

0 .2

0 .4

0 .6

0 .8

D a y s p o s tp a r tu m

Ha

pto

glo

bin

(m

g/m

l)

0

N o rm a l n = 7

T o x ic m e tr it is n = 1 3

M e tr it is n = 1 3

Sheldon & Dobson 2001 Vet Rec 148, 172-175

Uterine infection increases the concentrations of LPS, acute phase proteins, and prostaglandin E in blood

Herath et al 2009 Endocrinology 150:1912-1920

E. coli

Haptoglobin

Prostaglandin F

LPS

α1-acid glycoprotein

Prostaglandin E

IL-6 IL-10 IL-1β

TNFα TLR4 IL-1α

©2009 Herath et al Reprod Biol Endo

Innate immunity in the postpartum endometrium

Herath et al 2009 Reprod Biol Endocrinol 7: 55

Endometrial organ cultures accumulate inflammatory mediators in response to live E. coli and T. pyogenes

Borges et al 2012 AJRI 67, 526

IL-1β IL-6 IL-8

Endometrial cells mount inflammatory response to pathogen-associated molecules

IL-6 IL-8 Epithelium

Stroma

IL-6

(p

g/m

l)

0

6 0

1 2 0

1 8 0

2 4 0

* **

*

*

**

**

*

*

IL-6

(p

g/m

l)

0

5 0 0

1 0 0 0

1 5 0 0

2 0 0 0 **

*

*

*

IL-8

(p

g/m

l)

0

2 0

4 0

6 0

8 0

*

*

**

**

* *

IL-8

(p

g/m

l)0

3 0

6 0

9 0

1 2 0

*

*

*

* **

PAM

PAM

PAM

PAM

FSL-1

FSL-1

FSL-1

FSL-1

LPS

LPS

LPS

LPS

Turner et al 2014 Endocrinology 155, 1453

Endometrial cells detect pathogen-associated molecules

NFκB

0 min 45 min

LPS

vehicle

Cronin et al 2012 Biol Reprod 86, 51

Endometrial cells mount inflammatory response to pathogen-associated molecules via TLRs

TLR4 TLR2 TLR1 TLR6

Cronin et al 2012 Biol Reprod 86: 51 Turner et al 2014 Endocrinology 155, 1453-1465

si = siRNA

Primary stromal cells

LPS switches prostaglandin secretion in endometrial epithelial cells

Stromal cells Epithelial cells

0

50

100

150

200

*

*

Pros

tagl

andi

n (n

g/m

l) Prostaglandin E2

Prostaglandin F2a

Oxytocin LPS 1 mg/ml Oxytocin LPS 1 mg/ml0

50

100

150

200

*

*

Herath et al. 2009 Endocrinology 150:1912-1920

Pathogen-associated molecular patterns

Summary 1: endometrial cells generate inflammatory responses to pathogen-associated molecules

Danger model

Polly Matzinger

What factors might scale the inflammatory response in the endometrium?

DAMPs Damage-associated molecular patterns

Healey et al 2014 Scientific Reports 4: 7060

Host tissues and cells respond to IL-1α but not prototypical DAMPs, HMGB1 or hyaluronan

HA – hyaluronan – extracellular matrix DAMP HMGB1 – nuclear protein – cellular DAMP IL-1α - intracellular cytokine DAMP LPS – pathogen-associated molecule

Endometrium Blood

Healey et al 2014 Scientific Reports 4: 7060

Healy et al Scientific Reports 2014; 4: 7060

Endometrial cells sense and react to tissue damage during infection via interleukin 1α

Stroma

Epithelium

Supernatant

Supernatant

IL-1α scales endometritis when there is infection followed by cell damage

Healey et al 2014 Scientific Reports 4: 7060

Stroma

Epithelium

Countering infections requires a robust immune response and then resolution of the inflammation

What goes wrong in dairy cattle?

Inflammation

Days post partum

Chronic inflammation

Healthy

0 7 14 21 28

Reducing the availability of glucose blunts the innate immune response

Turner et al 2016 PLoS ONE 11: e0151416

0

2

4

6

8

IL-1

b (

pg

/mg

)

P < 0 .0 0 1

IL -1 b

0

5

1 0

1 5

2 0

IL-8

(p

g/m

g)

P < 0 .0 0 1

IL -8 C o n tro l

L P S

0

2 0 0

4 0 0

6 0 0

IL-6

(p

g/m

g)

P < 0 .0 0 1

IL -6

Endometrial organ culture

The sensor of cellular energy - AMPK - regulates the innate immune response

Ve

hic

le

AIC

AR

Co

mp

ou

nd

C

p h o s p o A M P K

to ta l A M P K

A IC A R

C o m p o u n d C

0 0 1 0 2 5 5 00

5

1 0

1 5

C o m p o u n d C (m M )

IL-1

b (

pg

/mg

)

P < 0 .0 0 1

0 0 1 0 2 5 5 00

1 0 0

2 0 0

3 0 0

C o m p o u n d C (m M )

IL-6

(p

g/m

g)

P < 0 .0 0 1

0 0 1 0 2 5 5 00

5

1 0

1 5

C o m p o u n d C (m M )

IL-8

(p

g/m

g)

P < 0 .0 0 1

0 0 2 5 0 5 0 0 1 0 0 00

5

1 0

1 5

A IC A R (m M )

IL-1

b (

pg

/mg

)

0 0 2 5 0 5 0 0 1 0 0 00

2 0 0

4 0 0

6 0 0

A IC A R (m M )

IL-6

(p

g/m

g)

P < 0 .0 0 1

0 0 2 5 0 5 0 0 1 0 0 00

1 0

2 0

3 0

4 0

A IC A R (m M )

IL-8

(p

g/m

g)

P < 0 .0 0 1

C o n tro l

L P S

Turner et al 2016 PLoS ONE 11: e0151416

Endometrial organ culture

Active resolution of inflammation

Phagocytosis and elimination of microbes

Sensing of vita-PAMPs

Inflammatory responses to

PAMPs

Sensing DAMPs from cell and tissue damage

Cell and tissue resilience counter

infections

Microbial virulence factors disrupt cells

Autocrine and paracrine regulation of innate immune response

Duration of microbial infection

Am

plitu

de o

f inf

lam

mat

ory

resp

onse

Nutrition and metabolism Ovarian hormones

Summary 2: The innate immune response reflects the severity of challenge and impact of the environment

Sheldon et al 2016 J Reprod Immunol

Sheldon et al 2002 Reproduction 123, 837 Herath et al 2007 Reproduction 134, 683

None Mild Moderate Severe

1

10

100

Folli

cual

r Flu

id L

PS (

ng/m

l)

Uterine inflammation

198

24

n = 7LPS

6 8 10 12 14 160

4

8

12

16

**

****

****

**** **

Folli

cle

diam

eter

(mm

) Normal uterus Uterine infection

6 8 10 12 14 160

1

2

3**

Oes

tradi

ol (p

g/m

l)

Days post partum

What impact does uterine infection have on ovarian function?

Conclusion: innate immunity is a key mechanism linking infection and infertility in the uterus and ovary

Sheldon IM, Price JC, Turner ML, Bromfield JJ, Cronin JJ 2014 Uterine infection and immunity in cattle. In “Reproduction in DomesticRuminants VIII”, pp 415-430. Editors: Juengel JJ, Miyamoto A, Price C, Reynolds LP, Smith MF, Webb R. Published by Context Products Ltd, Ashby de la Zouch, UK.